Local treatment of many diseases can occur by topical application of a drug that must be able to penetrate epithelial tissue. Adjacent epithelial cells are sealed by tight junctions, preventing the passage of most dissolved molecules from one side of the epithelial sheet to the other (Alberts et al., Molecular Biology of the Cell, 2nd ed.). Tight junctions are crucial for the formation and maintenance of the paracellular barrier and for cell polarity in simple epithelia and endothelia. They also play an important role at the blood brain barrier, where they control substances that leave or enter the brain. Large molecular weight drugs need to pass through these tissue barriers in order to get to their sites of action. In general, antibodies are too big to cross the tight junctions of epithelial cell layers.
As part of the body's normal activity, tight junctions selectively open and close in response to various signals both inside and outside of cells. This allows the passage of large molecules or even entire cells to across the tight junction barrier.
Mucosal administration of therapeutic compounds may offer certain advantages over injection and other modes of administration, for example in terms of convenience and speed of delivery, as well as by reducing or eliminating compliance problems and side effects that attend delivery by injection. However, mucosal delivery of biologically active agents is limited by mucosal barrier functions and other factors. For these reasons, mucosal drug administration typically requires larger amounts of drug than administration by injection. Other therapeutic compounds, including large molecule drugs, peptides and proteins, are often refractory to mucosal delivery.
The ability of drugs to permeate mucosal surfaces, unassisted by delivery-enhancing agents, appears to be related to a number of factors, including molecular size, lipid solubility, and ionization. Small molecules, less than about 300-1,000 daltons, are often capable of penetrating mucosal barriers, however, as molecular size increases, permeability decreases rapidly. Lipid-soluble compounds are generally more permeable through mucosal surfaces than are non-lipid-soluble molecules. Peptides and proteins are poorly lipid soluble, and hence exhibit poor absorption characteristics across mucosal surfaces.
US2006062758 provides compositions and methods that include a biologically active agent and a permeabilizing peptide effective to enhance mucosal delivery of the biologically active agent in a mammalian subject. The permeabilizing peptide reversibly enhances mucosal epithelial paracellular transport, typically by modulating epithelial junctional structure and/or physiology at a mucosal epithelial surface in the subject.
Peptides capable of modulating the function of epithelial tight junctions have been previously described (Johnson, P. H. and Quay, S. C., 2000). CA2379661 provides a paracellular drug delivery system comprising a Caludin-6 derived peptide. Claudins represent a super-family of integral membrane proteins located at the tight junctions and providing the barrier function.
Antibodies are powerful tools for biochemical and molecular biology research and are widely applied in medical diagnostics and therapy due to their ability to specifically bind to their antigen with high affinity. Typically, antibodies consist of two heavy and two light chains, which are covalently linked to each other via disulfide bonds. A highly variable domain comprising three complementary regions (CDRs) is positioned at the N-terminus of each chain. In concert, variable regions of the heavy and light chain determine antigen specificity of the antibody. Single chain antibodies (scFv) have been engineered by linking the DNA sequences encoding variable heavy (VH) and variable light (VL) domains with a spacer sequence coding for a flexible amino acid linker (Bird et al., 1988).
This format has the advantages over conventional full-length antibodies that an scFv is encoded by a single gene, mutations can be easily introduced, and the resulting scFv can be expressed in yeast and prokaryotic systems, which allow for rapid selection of specific high affinity binders to virtually any epitope by simple molecular biology. Due to their lack of effector function, scFv antibodies do not exert toxic effects via antibody-dependent or complement-dependent cell-mediated cytotoxicity (ADCC or CDCC, respectively), and unlike full-length antibodies, scFv antibodies show good tissue penetration abilities.
Many single chain antibodies (scFvs) have been generated against a multitude of different antigens, in particular because they can be easily selected for high binding capacity using techniques such as phage display or ribosome display. Moreover, scFv antibodies can be produced in microbial systems which are associated with fewer costs compared to the production of therapeutic full-length antibodies.
In addition to conventional extracellular and in vitro applications, scFvs have also been successfully used for intracellular applications (Worn et al. 2000; Auf der Maur et al. 2002; Stocks M R, 2004); hence, scFvs directed against intracellular antigens have been developed. In general, intracellular expression of functional scFvs is limited by their instability, insolubility, and tendency to form aggregates. For this reason, in vivo screening systems for scFv antibodies, which are particularly soluble and stable under reducing conditions typical for the intracellular environment (e.g. nucleus, cytoplasm) have been successfully developed using a so called “Quality Control” screen (WO0148017; Auf der Maur et al. (2001); Auf der Maur et al., 2004) and have led to the identification of particularly stable and soluble scFv framework sequences for such purposes (WO03097697). Furthermore, these frameworks show exceptional expression levels and enhanced stability and solubility properties also under natural, oxidizing conditions in the extracellular environment. Hence, these favourable biophysical and biochemical properties translate into favourable high production yields and enable these antibody fragments, once directed against specific antigens, to be applied locally and/or systemically as protein therapeutics in particular therapeutic areas.
For the use of antibodies in many therapeutic applications, in particular local applications, an important factor is the ability of the antibody to penetrate tissues, and in particular epithelial tissue barriers.
Local application is particularly desirable for the treatment of disorders that are manifested at a particular locus and do not require a systemic treatment, for example eye diseases.
Uveitis Anterior
Uveitis is an acute or chronic inflammation of the uvea with a prevalence of 30-40 per 100,000 (Lightman and Kok 2002). Uveitis is subdivided by location in uveitis anterior, intermediate or posterior. Uveitis anterior develops into uveitis posterior followed by complications such as cataracts, retinitis and even blindness, if left untreated (Kok and Lightman 2004). In <65 year olds there are as many legally blind individuals as a result from uveitis as diabetic retinopathy (Kok and Lightman 2004). Uveitis anterior, as the most common form of intra-ocular inflammatory diseases, is associated with histocompatibility locus A allele B27 (HLA-B27) in 50% of the cases (Power et al. 1998). Of these patients, only about half suffer from an additional systemic disease such as ankylosing spondylitis or chronic inflammatory bowel disease (El-Shabrawi and Hermann 2002). The treatment of uveitis is primarily aimed at controlling the inflammatory process (Kok and Lightman 2004). Currently, corticosteroids are the mainstay for therapy of uveitis (Kok and Lightman 2004). Importantly, local and systemic corticosteroid treatment significantly increases the risk of glaucoma and cataract, thus limiting its repeated use (El-Shabrawi and Hermann 2002). Other treatments including methotrexate, cyclosporine or azathioprine require a minimum of 6 weeks treatment to produce an effect, leaving patients with an enormous constraint of quality of life for a long period (El-Shabrawi and Hermann 2002, Dick et al. 1997).
From the above, a clearly defined medical need is obvious. Topical corticosteroids as the most common therapeutic option have significant side-effects, which in fact exacerbate the long term risk of blindness.
Recently TNFα concentrations of 15 pg/ml have been found in aqueous humor of uveitis patients, whereas the corresponding levels in healthy individuals were 0.56 pg/ml (Perez-Guijo et al. 2004). Several small clinical studies performed with systemically applied TNFα inhibitors report “immediate improvement” (El-Shabrawi and Hermann 2002) or “marked clinical improvement within days” (Murphy et al. 2004) or “within 2 weeks” (Joseph 2003) or “significant improvement after the first infliximab dose” (Benitez Del Castillo et al. 2004).
Thus, the concept of targeting TNFα is clinically well validated. However, safety concerns related to systemic application of TNFα inhibitors remain and would not justify their use in the significant fraction of uveitis patients who lack additional systemic disease manifestations.
Therefore, a topical TNFα inhibitor will fill a well-defined medical need, especially in patients with uveitis anterior. Due to their large molecular weight, the marketed TNFα inhibitors are not applicable topically (see Thiel et al. 2002).
Behçet's Disease
Behçet's disease is an idiopathic, multisystemic, chronic, and recurrent disease, classically characterized by episodic aggressive ocular inflammatory attacks, orogenital ulcers and skin lesions. In rare, severe cases of Behçet's disease, articular, audio-vestibular, thoracic gastrointestinal, cardiovascular, renal or CNS involvement may be observed in addition. The eye is the most commonly involved internal organ in Behçet's disease and is the leading cause of chronic morbidity in patients. Ocular disease consists of unilateral (20%) or bilateral (80%) iridocyclitis, hypopyon or panuveitis running a chronic and relapsing course. In general, initial exacerbations tend to be more anterior and unilateral, whereas subsequent attacks tend to involve vitreal cavity and posterior segment of the eye, becoming bilateral (Evereklioglu 2005). Severe uveitis is more commonly observed among patients form endemic regions such as Japanese and Turkish patients, affecting 70-90% of this population (Özen 1999; Tursen et al., 2003; Tugal-Tutkun et al., 2004; Yurdakul et al., 2004; Evereklioglu 2005). The risk of visual loss increases progressively, reaching one-fourth of the cases at 10 years. In addition, legal blindness is significant and eventually ensues in more than 50% of cases in countries with high prevalence and severity of the disease, such as Japan (Boyd et al., 2001; Evereklioglu 2005).
Behçet's disease exhibits a distinct geographic variation and is endemically higher particularly in Japan, Korea, Saudi-Arabia, Iran and Turkey as well as in the countries along the ancient “silk road”, including China and Israel (Bonfioli and Orefice 2005; Evereklioglu 2005). For example, Behçet's disease accounts for 20% of cases of uveitis in Japan and Turkey when compared with only 0.2% in the USA. In countries where the disease is endemic, it is more severe, with a higher frequency of ocular manifestations and complications and is more common in men, especially young male adults (Evereklioglu 2005). This peculiar epidemiology appears to be mediated by a combination of genetics (such as association with the HLA-B51 allele (Sakane et al., 1999; Verity et al., 1999; Evereklioglu 2005), infectious agents (Direskeneli 2001; Evereklioglu 2005) and environmental factors. The estimated prevalence of Behçet's disease is between 1:10,000 and 1:1000 in the Mediterranean countries, the Middle East, and the Far East. In Japan and Asian countries along the silk road, the prevalence is 13-30 per 100,000 and is highest in the northern parts of Japan; the highest overall prevalence with up to 400 per 100,000 is observed in certain parts of Turkey. There are approximately 15,000 people with Behçet's disease in the USA (Zierhut et al., 2003; Evereklioglu 2005).
Consequences of ophthalmic inflammatory attacks are the leading cause of chronic morbidity in Behçet's disease patients (Evereklioglu 2005). Treatment of Behçet's disease is symptomatic and empirical. As in other forms of uveitis, topical, periocular and systemic corticosteroids represent the mainstay of therapy in ocular Behçet's disease. However, the use of corticosteroid-based treatment modalities in the patients is limited by their significant side-effect profile. In addition, corticosteroids rarely induce complete remissions in ocular Behçet's disease and a significant fraction of patients develops steroid-resistant disease over time (Evereklioglu 2005). In the course of the disease, treatments frequently comprise immunosuppressive agents such as azathioprine, methotrexate and cyclosporine A. However, as these agents are associated with critical safety issues as well, there is a well-expressed medical need for an efficient and safe novel treatment modality in this indication.
Besides recent epidemiological findings that suggest polymorphic variations in TNFα to be associated with the severity of Behçet's disease (Verity et al., 1999b), there exists a broad variety of case reports and small clinical trials describing the use of infliximab in ocular Behçet's disease (Ohno et al., 2004; Wechsler et al., 2004; Giansanti et al., 2004; Lanthier et al., 2005; Tugal-Tutkun et al., 2005; Lindstedt et al., 2005). In fact, all these studies report rapid and complete remission of ocular Behçet's disease, even in patients resistant to conventional therapy (Tugal-Tutkun et al., 2005). However, the frequency and severity of adverse events in infliximab-treated uveitis patients is unexpectedly high in some studies, thus limiting the potential of systemically applied TNFα antagonists for treatment of this disease (Rosenbaum 2004; Suhler et al., 2005).
The clinical validation of TNFα as a highly attractive drug target in ocular Behçet's disease (Ohno et al., 2004; Wechsler et al., 2004; Giansanti et al., 2004; Lanthier et al., 2005; Tugal-Tutkun et al., 2005; Lindstedt et al., 2005) and the apparent safety concerns with systemic TNFα suppression in uveitis patients (Rosenbaum 2004; Suhler et al., 2005), reveals that there is a need for the development of a topically applicable TNFα antagonist for ocular Behçet's disease, especially for patients with predominant ocular symptoms.
Due to their good tissue penetration abilities and their rapid renal clearance, scFv antibodies are preferred for local applications. Besides charge, hydropathicity and molecular weight, properties such as solubility, aggregation tendency and thermal stability influence the ability of a molecule to penetrate through tissue barriers. For example, a highly soluble antibody fragment may not be able to penetrate epithelial barriers if it forms aggregates at physiological temperature around 37° C. Mutation of a single amino acid residue in an scFv framework may on the one hand improve its solubility at ambient temperature, and this mutation may alter thermal stability and therefore lead to partial unfolding and aggregation at 37° C. Such aggregates, due to their higher molecular weight, are no longer able to pass tissue barriers.
Because tissue penetration is an important factor for efficient drug delivery, in particular in local applications, there is a need for therapeutic antibodies, in particular scFv antibodies with improved tissue penetration abilities besides the otherwise desirable characteristics of high stability and low antigenicity. WO0040262 discloses antibody fragments, e.g. scFvs, as pharmaceuticals or diagnostic tools to treat or diagnose, respectively, ocular disorders. Eye penetration experiments are done at concentrations of 0.2 to 0.25 mg/ml scFv. It was shown that an scFv could penetrate the epithelial barrier of the cornea at a very low rate in the absence, and at higher rates in the presence of penetration enhancers. Since penetration enhancers can have cytotoxic effects or cause epithelial alterations, there is a need for alternative and/or improved methods for the treatment of ocular diseases by scFvs and fragments thereof. In particular, antibodies are needed for controlled therapy by local administration with a low degree of side effects, which can be administered at a relatively high concentration.
All publications and references cited herein are hereby incorporated by reference in their entirety.